Acute myeloid leukemia (AML) represents ~90% of all acute leukemias in adults with only 25% survival at 5 years. The main cause of death is the uncontrolled proliferation of hematopoietic progenitor blast cells of myeloid lineage. Despite numerous experimental strategies, the standard of care for this cancer has not significantly changed or improved over 30 years. AML is a genetically heterogeneous disease, which renders the design of tailored therapy challenging. Aggressive chemotherapy with cytarabine and an anthracycline is the most efficient treatment that reduces blast counts, induces stable remission and prolongs survival. Unfortunately, for elderly, immunocompromised patients, aggressive chemotherapy causes severe morbidity and mortality, and could be fatal. There is a broad unmet need for therapeutic approaches that can decrease the systemic toxicity of chemotherapy. The objective of this proposal is to develop red blood cell-based therapy for AML. We previously described a rapid and efficient modification (painting) of the membrane of red blood cells (RBCs) with antibodies and small molecules and demonstrated that these cells specifically can deplete circulating cells in animal lymphoma models. Pharmacokinetic simulations suggest that a 3-day half-life of painted RBCs in mice scales up to 30 days of lifespan in humans, suggesting that RBC administration has great potential to be developed into a highly practical approach. Supported by strong preliminary data, the objective of this proposal is to develop painted RBCs for efficient ablation of blasts in a preclinical model of AML. The mechanism of ablation involves capturing blasts in the circulation and subsequently eliminating them by liver macrophages. Since painted RBCs efficiently accumulate in the spleen and bone marrow (the main sites of blast residence), we will paint RBCs with a lipid prodrug of idarubicin in order to ablate blasts in these organs, while lowering systemic dose and toxicity. We developed the following Specific Aims: 1) Demonstrate efficient in vitro targeting of AML blasts by RBCs painted with antibodies specific for myeloid markers CD33 and CD123; 2) Investigate factors that control in vivo ablation efficiency of antibody-painted RBCs; 3) Synthesize idarubicin prodrugs for painting the RBC membrane and test in vitro cytotoxicity; 4) Determine the in vivo therapeutic effect of antibody- and drug-painted RBCs in vivo in preclinical mouse models of AML. If successful, we presume that subsequent clinical studies will be feasible. Painting of RBCs (autologous or donor-sourced) is a highly practical and effective procedure that can be carried out in a clinical setting. RBC-mediated ablation of leukemic blasts is a novel approach that can decrease the amount of chemotherapy required to achieve stable remission while decreasing treatment-associated morbidity and mortality in patients.